Characterization of antiviral activity of green tea extract and applications for improving food safety

Los norovirus, el virus de la hepatitis A y el virus de la hepatitis E son algunos de los principales riesgos sanitarios relacionados con el consumo de alimentos teniendo un alto impacto en la seguridad alimentaria y siendo responsables de diversas patologías en los consumidores (desde gastroenteritis leves hasta patologías de carácter serio como pueden ser hepatitis o incluso meningitis o encefalitis). Debido a que los virus entéricos se transmiten principalmente por la vía fecal-oral, su presencia puede darse en alimentos que se hayan contaminado de forma directa con material fecal o mediante agua contaminada. Los alimentos mayoritariamente implicados en este tipo de infecciones son los moluscos bivalvos, vegetales y ensaladas, bayas y productos listos para consumir los cuales se han contaminados por una incorrecta manipulación después de su preparación o cocinado. Como consecuencia del incremento de brotes de transmisión alimentaria, organismos internacionales como la Organización Mundial de la Salud (OMS) y la Autoridad Sanitaria de Seguridad Alimentaria (EFSA), proponen evaluar la eficacia de procesos alimentarios para la inactivación de virus. Además, la OMS está promoviendo el desarrollo de métodos alternativos para la descontaminación de alimentos. Con esta finalidad, la presente tesis doctoral se ha centrado en el efecto del extracto de té verde (GTE) frente a virus entéricos humanos y su uso en alimentos o en superficies de contacto alimentario con el fin de mejorar la seguridad alimentaria. Inicialmente, la actividad virucida y la caracterización del GTE se evaluó frente al norovirus murino (MNV), un virus modelo de los norovirus humanos, y el HAV a diferentes temperaturas, tiempos de contacto y condiciones de pH, mostrando efectividad frente a estos dos virus en función del pH y observándose mayores reducciones en condiciones alcalinas y neutras. Al mismo tiempo, diferentes concentraciones del GTE (0,5 y 5 mg/mL) se mezclaron a partes iguales con la suspensión viral y fueron incubadas durante 2 y 16 horas a 4, 25 y 37°C. Los resultados mostraron la completa inactivación de ambos virus después de tratamientos a 37°C durante 16 horas y a 25ºC para el HAV. En posteriores ensayos se observó que el GTE mejoraba su actividad cuando se preparaba 24 horas antes de su aplicación (aged-GTE). Por otro lado, los análisis mediante HPLC/MS demostraron que la mejora de la actividad antiviral estaba relacionada con los cambios en la composición química del GTE debido a la formación de catequinas que se producía durante el periodo de 24 horas, principalmente a la epigalocatequina galato (EGCG) suponiendo alrededor de un 40-50% de la composición del GTE. Por otra parte, el efecto del GTE sobre norovirus humanos fue evaluado indirectamente mediante partículas pseudovíricas (VLPs) y ensayos con mucina gástrica porcina (PGM) unida a la técnica ELISA y microscopía electrónica de transmisión además de analizar dicho efecto en suspensiones de norovirus humanos por la técnica in situ capture-RT-qPCR. Además, se realizaron ensayos de viabilidad por RT-qPCR tratando suspensiones de norovirus con EGCG combinadas con PMAxx como agente intercalante. Los resultados obtenidos mostraron reducciones cercanas al 50% en la capacidad de unión a la PGM. Sin embargo, los resultados de PCR de viabilidad indicaron que el tratamiento con ECGC no afecta a la cápside viral de manera considerable. Con el objetivo de utilizar el GTE para mejorar la seguridad alimentaria, en el marco de esta tesis se evaluó su uso en diversas aplicaciones alimentarias. Inicialmente, se evaluó la estabilidad del GTE en condiciones gástricas simuladas (saliva, gástrica e intestinal) para dilucidar su eficacia como compuesto antiviral terapéutico frente a virus entéricos después de la ingesta. Estos ensayos demostraron que los niveles de MNV se redujeron por debajo del límite de detección mientras que los del HAV se redujeron en 2 órdenes logarítmicos. Además, el GTE fue incorporado a diferentes bebidas (zumos de naranja y manzana, horchata y leche) que se trataron con GTE XXX. Los resultados mostraron reducciones por debajo del límite de detección para MNV en zumo de manzana, mientras que para el HAV los mejores resultados se obtuvieron en zumo de manzana, con más de 2 ordenes logarítmicos de reducción cuando se trató con GTE a 5 mg/mL durante 16 horas a 37⁰C. Cuando las bebidas se trataron mediante tratamientos térmicos moderados combinados con la adición de GTE se observó un aumento de la inactivación del MNV. En cambio, este efecto sinérgico no se observó para el HAV. Posteriormente se evaluó el GTE como higienizante natural para la desinfección de superficies de contacto alimentario y vegetales. Estos ensayos se realizaron según la norma ISO 13697:2001, inoculando suspensiones de MNV y HAV sobre superficies de acero inoxidable y vidrio y añadiendo posteriormente la solución de GTE que se dejó actuar durante de 15 y 30 minutos. Una metodología similar se llevó acabo en ensayos de desinfección en hojas de lechuga y espinacas frescas. Los resultados obtenidos mostraron reducciones de 1,5 órdenes logarítmicos e inactivación total para MNV y HAV en acero inoxidable y vidrio tratados con GTE (10 mg/mL), respectivamente. Resultados similares se obtuvieron en los tratamientos de lechuga y espinacas bajo las mismas condiciones experimentales. Por último, el GTE fue incorporado a recubrimientos comestibles con el objetivo de minimizar la contaminación de virus en frutos tipo baya. La adición se efectuó a matrices de alginato/oleico y diferentes carragenatos aplicándose sobre arándanos y frambuesas resultando en la mejora de la actividad antiviral tanto a temperatura ambiente como de refrigeración, siendo algo más efectivo para MNV. Finalmente, en base a los resultados de la presente tesis, se puede concluir que el GTE es una opción natural y de bajo coste para la mejora de la seguridad alimentaria frente a virus entéricos.Norovirus, hepatitis A virus (HAV) and more recently hepatitis E virus (HEV) are some of the main health risks associated with food consumption having a high impact on food safety and being responsible for diverse pathologies in consumers (from moderate gastroenteritis to more serious pathologies such as hepatitis or even meningitis or encephalitis). Because enteric viruses are mainly transmitted by the faecal-oral route, their presence can occur in food which has been directly contaminated with faecal material or contaminated water. The main foodstuffs involved in foodborne infections are mollusc bivalves, vegetables and salads, berries, and ready-to-eat food which have been contaminated by improper handling after their preparation or cooking. As a consequence of the increasing number of foodborne outbreaks, international organisms, such as the World Health Organization (WHO) or the European Food Safety Authority (EFSA), propose studies on the efficacy of food processes for virus inactivation. Moreover, the WHO is promoting the development of alternative methods for the decontamination of food. To this end, the present doctoral thesis has focused on the effect of green tea extract (GTE) against human enteric viruses and its potential application in food products or food contact surfaces to enhance food safety. Initially, the antiviral activity and characterization of GTE was evaluated on murine norovirus (MNV), a human norovirus surrogate, and HAV with different temperatures, exposure times and pH conditions showing effectiveness against both viruses depending on pH with higher reductions observed in alkaline and neutral conditions. At the same time, different concentrations of GTE (0.5 and 5 mg/mL) were mixed with viral suspensions and incubated for 2 or 16h (overnight) at 4, 25 and 37°C. Complete viral inactivation was achieved after overnight exposure at 37°C for both viruses and also at 25°C for HAV. In following assays, it was observed that GTE enhanced its antiviral activity when the GTE solution was prepared 24h before its application (aged-GTE). Additional HPLC/MS analyses demonstrated that the enhanced antiviral activity was related to changes in the chemical composition resulting from the formation of catechin derivatives during storage, mainly epigallocatechin gallate (EGCG) that accounted for around 40-50% of GTE composition. Furthermore, norovirus inactivation by GTE was assessed indirectly by testing the effect of aged-GTE on virus-like particles (VLPs) of human norovirus by a porcine gastric mucine (PGM) binding ELISA and transmission electron microscopy (TEM), and on human norovirus suspensions by an in situ capture-RT-qPCR method. Viability RT-qPCR was also evaluated by treating norovirus with EGCG in combination with PMAxx as intercalating dye. Results displayed a significant reduction of close to 50% in the binding to PGM. However, results from viability RT-qPCR indicated that EGCG did not dramatically affect the viral capsid. With the aim to apply GTE to enhance food safety, the potential application of aged-GTE was evaluated in different scenarios. Firstly, the stability of GTE was evaluated in simulated gastric conditions (salivary, gastric and intestinal) to shed light on its capacity as a therapeutical antiviral compound to fight enteric viruses inside the organism. MNV titers decreased to undetectable levels while HAV infectivity reduced by 2 log. Moreover, aged-GTE was incorporated into different contaminated beverages (orange and apple juice, horchata and milk). Results showed a reduction of infectious titers to undetectable limits for MNV in apple juice, while the best HAV reductions were reported in apple juice with a more than 2 log decrease when treated at 5 mg/mL of aged-GTE at 37⁰C for 16 hours. When food models were exposed to moderate heat treatments combined with aged-GTE, increased MNV inactivation was reported. Nevertheless, no such synergistic effect was observed for HAV. Subsequent assays were on track to elucidate the antiviral behaviour of GTE as natural sanitizers for food contact surfaces and washing solution for vegetables. Based on the ISO 13697:2001 standard, suspensions of MNV and HAV were spotted and dried on stainless steel and glass food contact surfaces. Then, aged-GTE was pipetted above the viral inoculums for 15 and 30 min and finally recovered. A similar methodology was performed in the sanitation tests for fresh lettuce and spinach leaves. Results reported a 1.5 log reduction and complete inactivation was observed for MNV and HAV on stainless steel and glass surfaces that had been treated with 10 mg/mL of GTE for 30 min. In lettuce and spinach, MNV and HAV titers were reduced by more than 1.5 log after a 30 min treatment. Finally, GTE was incorporated into edible coatings with the goal of controlling virus contamination in berries. The addition of aged-GTE to carrageenan and alginate/oleic matrices applied on blueberries and raspberries improved the antiviral activity at both refrigerated and ambient temperatures, being slightly more effective in the case of MNV. Lastly, supported by the results of the present thesis, it can be concluding that GTE is a natural and inexpensive option to improve viral food safety

Rapid Selective Detection of Potentially Infectious Porcine Epidemic Diarrhea Coronavirus Exposed to Heat Treatments Using Viability RT-qPCR.

Coronaviruses (CoVs) cause severe respiratory, enteric, and systemic infections in a wide range of hosts, including humans and animals. Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family, is the etiological agent of porcine epidemic diarrhea (PED), a highly contagious intestinal disease affecting pigs of all ages. In this study, we optimized a viability real-time reverse transcriptase polymerase chain reaction (RT-qPCR) for the selective detection of infectious and heat-inactivated PEDV. PEMAX , EMA , and PMAxx photoactivable dyes along with PtCl4 and CDDP platinum compounds were screened as viability markers using two RT-qPCR assays: firstly, on PEDV purified RNA, and secondly on infectious and thermally inactivated virus suspensions. Furthermore, PMAxx pretreatment matched the thermal inactivation pattern obtained by cell culture better than other viability markers. Finally, we further optimized the pretreatment by coupling viability markers with Triton X-100 in inoculated serum resulting in a better estimation of PEDV infectivity than RT-qPCR alone. Our study has provided a rapid analytical tool based on viability RT-qPCR to infer PEDV infectivity with potential application for feed and feed ingredients monitoring in swine industry. This development would allow for greater accuracy in epidemiological surveys and outbreak investigations

Virus entéricos humanos en alimentos: detección y métodos de inactivación

Los principales patógenos víricos que podemos ad­quirir ingiriendo alimentos contaminados son los norovirus, el virus de la hepatitis A y el virus de la hepatitis E que se propagan principalmente a través de la vía fecal oral. En los últimos años, la incidencia de brotes de transmisión alimentaria causados por estos patógenos ha experimentado un aumento considerable, en parte debido al comercio globalizado y a los cambios en los hábitos de consumo. Las matrices alimentarias que mayor riesgo representan para el consumidor son los moluscos bivalvos, ve­getales de IV gama, frutas tipo baya y platos listos para comer. Actualmente las técnicas moleculares son las más habituales para la detección de estos patógenos en alimentos, aunque toda­vía existen dudas acerca del significado de la presencia de estos genomas víricos en términos de seguridad alimentaria. La infec­tividad de estos patógenos en alimentos viene también determi­nada por su elevada persistencia ambiental y por su resistencia a los tratamientos aplicados para la conservación de los alimentos

High pressure treatment and green tea extract synergistically control enteric virus contamination in beverages

Consumers are driving food production toward the use of natural preservatives and minimal processing technologies. Green tea extract (GTE) at low concentration could be combined with high pressure processing (HPP) for reduced treatment times and quality impact on foods in a hurdle concept for synergistic effects on foodborne viral pathogens, specifically human norovirus and hepatitis A virus (HAV). Viral inactivation by HPP (at 300, 400, and 500 MPa for 5 min) combined with 3.3 mg/mL aged-GTE was initially evaluated in buffer (PBS) against murine norovirus (MNV), a culturable human norovirus surrogate, and HAV. Furthermore, human norovirus inactivation was evaluated by the novel human intestinal enteroid system (HIE) and a capsid integrity binding assay (ISC-RT-qPCR). HPP treatment completely inhibits human norovirus GII.4 infectivity when applied at 500 MPa alone and at 400 MPa combined with aged-GTE. Additional experiments investigated the reduction of MNV and HAV infectivity in apple and horchata juices exposed to combined aged-GTE and HPP treatments. Results demonstrated that the addition of aged-GTE to the juices exposed to HPP significantly inactivated MNV and HAV at reduced holding pressure time. This synergistic effect of aged-GTE combined with HPP treatments represents a hurdle technology that could be exploited as a control measure to improve the food safety of beverages

Spatial and temporal distribution of SARS-CoV-2 diversity circulating in wastewater.

Wastewater-based epidemiology (WBE) has proven to be an effective tool for epidemiological surveillance of SARS-CoV-2 during the current COVID-19 pandemic. Furthermore, combining WBE together with high-throughput sequencing techniques can be useful for the analysis of SARS-CoV-2 viral diversity present in a given sample. The present study focuses on the genomic analysis of SARS-CoV-2 in 76 sewage samples collected during the three epidemiological waves that occurred in Spain from 14 wastewater treatment plants distributed throughout the country. The results obtained demonstrate that the metagenomic analysis of SARS-CoV-2 in wastewater allows the detection of mutations that define the B.1.1.7 lineage and the ability of the technique to anticipate the detection of certain mutations before they are detected in clinical samples. The study proves the usefulness of sewage sequencing to track Variants of Concern that can complement clinical testing to help in decision-making and in the analysis of the evolution of the pandemic

Platinum chloride-based viability RT-qPCR for SARS-CoV-2 detection in complex samples.

Isolation, contact tracing and restrictions on social movement are being globally implemented to prevent and control onward spread of SARS-CoV-2, even though the infection risk modelled on RNA detection by RT-qPCR remains biased as viral shedding and infectivity are not discerned. Thus, we aimed to develop a rapid viability RT-qPCR procedure to infer SARS-CoV-2 infectivity in clinical specimens and environmental samples. We screened monoazide dyes and platinum compounds as viability molecular markers on five SARS-CoV-2 RNA targets. A platinum chloride-based viability RT-qPCR was then optimized using genomic RNA, and inactivated SARS-CoV-2 particles inoculated in buffer, stool, and urine. Our results were finally validated in nasopharyngeal swabs from persons who tested positive for COVID-19 and in wastewater samples positive for SARS-CoV-2 RNA. We established a rapid viability RT-qPCR that selectively detects potentially infectious SARS-CoV-2 particles in complex matrices. In particular, the confirmed positivity of nasopharyngeal swabs following the viability procedure suggests their potential infectivity, while the complete prevention of amplification in wastewater indicated either non-infectious particles or free RNA. The viability RT-qPCR approach provides a more accurate ascertainment of the infectious viruses detection and it may complement analyses to foster risk-based investigations for the prevention and control of new or re-occurring outbreaks with a broad application spectrum

Evaluation of two different concentration methods for surveillance of human viruses in sewage and their effects on SARS-CoV-2 sequencing

During the current COVID-19 pandemic, wastewater-based epidemiology (WBE) emerged as a reliable strategy both as a surveillance method and a way to provide an overview of the SARS-CoV-2 variants circulating among the population. Our objective was to compare two different concentration methods, a well-established aluminum-based procedure (AP) and the commercially available Maxwell® RSC Enviro Wastewater TNA Kit (TNA) for human enteric virus, viral indicators and SARS-CoV-2 surveillance. Additionally, both concentration methods were analyzed for their impact on viral infectivity, and nucleic acids obtained from each method were also evaluated by massive sequencing for SARS-CoV-2. The percentage of SARS-CoV-2 positive samples using the AP method accounted to 100 %, 83.3 %, and 33.3 % depending on the target region while 100 % positivity for these same three target regions was reported using the TNA procedure. The concentrations of norovirus GI, norovirus GII and HEV using the TNA method were significantly greater than for the AP method while no differences were reported for rotavirus, astrovirus, crAssphage and PMMoV. Furthermore, TNA kit in combination with the Artic v4 primer scheme yields the best SARS-CoV-2 sequencing results. Regarding impact on infectivity, the concentration method used by the TNA kit showed near-complete lysis of viruses. Our results suggest that although the performance of the TNA kit was higher than that of the aluminum procedure, both methods are suitable for the analysis of enveloped and non-enveloped viruses in wastewater by molecular methods

Sustainable bioactive pectin-based films to improve fruit safety via a circular economy approach.

This work reports on the valorisation of persimmon (Diospyros kaki Thunb.) for the development of food-grade antiviral coatings against major viral foodborne pathogens, human noroviruses (NoVs) and hepatitis A virus (HAV). Initially, the antiviral activity of polyphenol-rich pectin extracts with abundant non-covalent interactions (PPN), pectin extracts enriched with intact pectin-polyphenol ester and O-glycosyl bonds (PPC) and hydro-ethanolic polyphenol-rich extracts (EPE) was compared. Higher viral reductions were found for the pectin extracts rich in polyphenols, mainly in those containing covalent pectin-polyphenol interactions. This specific extract was mixed with commercial citrus pectin (CP) to develop active edible films. Dry films were analysed in terms of their optical, morphological, mechanical and barrier properties. Addition of the bioactive pectin persimmon extract resulted in more coloured films with lower transparency. The presence of covalently-linked polyphenols gave rise to stiffer films, with lower sorption capacity and more hydrophobic nature. The infectivity of MNV and HAV on fresh blueberries after the coating treatments was reduced by approximately 4.28 and 2.38 log, respectively, after overnight incubation, as compared to the controls, when 10% PPC was incorporated into the film. Higher amounts of PPC did not significantly improve the antiviral activity and a complete inactivation for both viruses was observed after 4 days of storage at 25 °C. This paper highlights the potential of persimmon discards as a cheap source of food-grade antiviral coatings with improved physicochemical properties as compared to commercial citrus pectin

Bioactive extracts from persimmon waste: influence of extraction conditions and ripeness.

In this work, a bioactive persimmon extract was produced from discarded fruits. A central composite design was used to evaluate the effect of different extraction parameters and ripeness stages of persimmon fruits on the total phenolic content and antioxidant activity of the resulting extracts. Significantly greater phenolic contents were obtained from immature persimmon (IP) fruits. The optimum IP extract with the conditions set by the experimental design was industrially up-scaled and its composition and functional properties were evaluated and compared with those obtained under lab-scale conditions. Both extracts contained significant protein (>20%) and phenolic contents (∼11-27 mg GA/g dry extract) and displayed significant antiviral activity against murine norovirus and hepatitis A virus. Moreover, the extract showed no toxicity and significantly reduced the fat content and the cellular ageing of Caenorhabditis elegans (C. elegans) without affecting the worm development. These effects were mediated by down-regulation of fat-7, suggesting an anti-lipogenic activity of this extract

Antiviral capacity of sanitizers against infectious viruses in process water from the produce industry under batch and continuous conditions

The presence of human enteric viruses in produce has extensively been reported. However, the significance of the quality of process water (PW) used by the produce industry and the viral inactivation capacity of water disinfection agents used to maintain the microbiological quality of PW has received limited attention. This study evaluates the antiviral disinfection efficacy of chlorine, chlorine dioxide (ClO2) and peracetic acid (PAA) at recommended operational limits in PW using hepatitis A virus (HAV), the cultivable norovirus surrogate, murine norovirus (MNV-1), and MS2 coliphages. Defined commodity representative crops (baby leaves, bell peppers, and the vegetable mix of tomatoes, cucumbers, peppers, and onions) associated with specific water-based processes were studied. Two systems classified as either batch or continuous system were used. The continuous system allows the continuously entrance of sanitizer solution and organic matter added to the washing tank to simulate the conditions of an industry wash tank. Batch scale experiments showed that 20 mg/L chlorine and 3 mg/L chlorine dioxide completely inactivated MNV-1 and MS2 (mean of 5 log) after 1 min contact time regardless of the PW type. However, the infectivity of HAV was reduced only by less than 2 log after 1 min for chlorine and chlorine dioxide and the complete inactivation was not observed even after 10 min. On the contrary, residual viral infectivity/viability of HAV, MNV-1 and MS2 was observed for PAA in the three types of PW. The inactivation kinetic models for MS2 coliphages were developed based on the data obtained under the continuous system comparing the three types of PW. Chlorine (5 mg/L) and chlorine dioxide (2-3 mg/L) avoided the accumulation of MS2 below the detection limit while PAA (80 mg/L) was unable to prevent it independently of the type of PW. In summary, in the washing operation, it is a key objective to reach virus inactivation through the selection of the most effective sanitizer by guaranteeing that sufficient concentration and contact times prevent the risk of viral cross-contamination